This invention relates to a screening method for the identification of potential fungicides, based upon use of yeast strains defective in ergosterol biosynthesis.
Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, except that ergosterol has a methyl group and two double bonds not present in cholesterol. In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle. Yeast cells can take up cholesterol and decrease their requirement for ergosterol to very low levels, but cholesterol alone cannot completely substitute for ergosterol (Gaber, R. F., et al., Mol. Cell. Biol. 9: 3447-3456 (1989)).
Though sterol biosynthesis in different organisms have many common steps, the biosynthesis of ergosterol involves steps distinct from cholesterol biosynthesis. These occur after ring formation from squalene, in the transformation of lanosterol to other sterols. One such step is sterol methylation at position 24 in the ergosterol biosynthetic pathway. The enzyme S-adenosylmethionine:xcex9424-sterol-C-methyl transferase is responsible for the alkylation. The ERG6 gene encoding this enzyme has been cloned and found to be not essential for growth, but erg6 mutants lacked normal membrane function (Gaber, et al., cited above, and McCammon, M. T., et al., J. Bact. 157: 475-483 (1984)).
Another enzyme unique to ergosterol biosynthesis is sterol xcex9422 desaturase, which catalyzes the desaturation of ergosta-5,7-dien-3-beta-ol, forming a double bond in ergosterol between carbons at positions 22 and 23 that are not present in cholesterol. This appears to be catalyzed by a cytochrome P450 (Hata, S., et al., Biochem. Biophys. Res. Com. 116: 162-166 (1983)).
The term xe2x80x9ccytochrome P450xe2x80x9d is a trivial name for a class of cytochromes that includes a number of heme proteins that have a characteristic absorption maximum at 450 nm when combined with Co in the reduced state (xe2x80x98Pxe2x80x99 denotes pigment; hence, the name). These cytochromes occur in many animal tissues, plants and microorganisms and catalyze the monooxygenation of a vast variety of hydrophobic substances including lipophilic endogenous compounds and xenobiotics, serving as oxygenating catalysts in the presence of one or more electron-transfer proteins or redox enzymes. These cytochromes act in concert with a reductase, a flavoprotein that serves as an electron-transfer enzyme from NADPH to cytochrome P450.
A second cytochrome P450 implicated in the ergosterol biosynthetic pathway has a counterpart in cholesterol biosynthesis. This is lanosterol 14-xcex1-demethylase, which in yeast catalyzes the oxidative removal of a methyl group at carbon position 14 of lanosterol during ergosterol biosynthesis, and the mammalian orthologue catalyzes the identical reaction in cholesterol biosynthesis (Turi, T. G., and Loper, J. C., J. Biol. Chem. 276: 2046-2056 (1992)). Though in higher organisms the diversity of oxidative reactions and broad substrate specificity of the cytochrome P450s are due primarily to the presence of multiple P450s encoded by a gene superfamily that may contain more than 100 genes (Kalb, V. F. et al., DNA 6: 529537 (1987)), yeast has been suggested as a model for cytochrome-P450 systems in all eukaryotes (Kalb, V. F., et al., Gene 45: 237-245 (1986)). Active enzyme from a mammalian cytochrome P450 clone has been expressed in Saccharomyces cerevisiae (Oeda, K., et al., DNA 4: 203-210 (1985)).
Cytochrome P450 reductases from a variety of sources have been isolated and compared, and the baker""s yeast (Saccharomyces cerevisiae) cytochrome P450 reductase gene has been cloned and characterized (Yabusaki, Y., et al., J. Biochem. 103: 1004-1010 (1988)). Studies of yeast mutants lacking the cytochrome P450 reductase gene showed that they were viable (Sutter, T. R., and Loper, J. C., Blochem. Biophys. Res. Com. 160: 1257-1266 (1989)). However, the cytochrome P450 reductase yeast mutants were 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-xcex1-demethylase mentioned above, indicating that the mutants were partially defective. No evidence that these mutants had a second cytochrome P450 reductase gene could be found, so apparently an alternate pathway provides for the functions of this reductase in S. cerevisiae. 
It is an object of the invention to provide a screening test for the identification of agents exhibiting potential fungicidal activity for a wide variety of agricultural, medical, and veterinary uses, and for the identification of potential antihypercholesterolemic agents.
It is a further and more specific object of this invention to identify agents that inhibit cytochrome P450 reductase or other enzymes or electron-transfer proteins associated with cytochrome P450 reductase in the ergosterol biosynthetic pathway.
These and other objects are accomplished by the present invention, which provides a method for the identification of agents which inhibit cytochrome P450 reductase involved in ergosterol biosynthesis. The method is a screening test whereby test samples are simultaneously incubated in a culture of a yeast strain supersensitive to inhibitors of cytochrome P450 reductase enzymes or electron-transfer proteins and a second yeast strain exhibiting less sensitivity to these compounds. Agents that are positive in the test inhibit growth more in the supersensitive strain than in the less sensitive strain.
In the practice of this invention""s method for screening for the presence or absence of inhibition of cytochrome P450 reductase or cytochrome P450 reductase related enzymes or electron-transfer proteins (cytochrome P450s) by a test sample, the test sample is added to a culture or culture area having a Saccharomyces cerevisiae strain having a disrupted cytochrome P450 reductase gene. At the same time, the test sample is added to a control culture or culture area having a Saccharomyces cerevisiae strain exhibiting enhanced membrane permeability. The cultures are incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth, ordinarily monitored in corresponding cultures or culture areas of the strains containing no test sample. The extent of inhibition of growth in the culture or culture area containing the cytochrome P450 reductase mutant is then compared with the extent of inhibition in the culture or culture area containing the strain having enhanced membrane permeability. The presence of inhibition of-cytochrome P450 reductase or cytochrome P450 reductase related enzymes or electron-transfer proteins is determined by observation of whether culture growth inhibition in the reductase mutant exceeds that in the permeable mutant.
In a preferred screening test, a Saccharomyces cerevisiae strain having a disrupted cytochrome P450 reductase gene is grown in culture in the presence of test samples. At the same time, a S. cerevisiae strain having an erg6 mutation is grown in culture with the same test samples. Potentially active agents are identified by the observation of enhanced inhibition of the cytochrome P450 reductase disrupted strain over the erg6 strain. A positive control is employed to assist in the identification of potential agents. In these embodiments, a known inhibitor of cytochrome P450 is used, such as, for example, the lanosterol 14-xcex1-demethylase inhibitors ketoconazole, miconazole, dinaconazole or econazole. Controls are added to both cultures or culture areas, and relative inhibition is compared with that observed with the test samples.
In a particularly preferred embodiment, the cytochrome P450 reductase disrupted strain and the erg 6 yeast strain are grown in a solidified media in a plate or dish, so that test samples and positive controls can be observed visually and simultaneously as regions of the same culture. Actives produce a much larger zone (xcx9cxe2x89xa78 mm) around test samples grown in a lawn of the reductase deleted strain than in a lawn of the erg6 strain.
This screening method is based upon the finding that a pair of Saccharomyces cerevisiae strains exhibiting defective ergosterol biosynthesis are useful for a screening assay for inhibitors of cytochrome P450 reductase, and cytochrome P450 reductase related enzymes or electron-transfer proteins (i.e., cytochrome P450 species). One strain is supersensitive to inhibitors of cytochrome P450 species and the second strain exhibits less sensitivity to these compounds.
Saccharomyces cerevisiae strains supersensitive to inhibitors of cytochrome P450 species include strains having a disrupted or deleted cytochrome P450 reductase gene. Baker""s yeast is easily manipulated genetically, and, as has been discussed, the NADPH-cytochrome P450 reductase yeast gene has been well studied, with the primary structure known (Yabusaki, Y., et al., cited above). Disruption of the gene can be effected using published procedures (summarized in Ausubel, F. M., et al., eds., Current Protocols in Molecular Biology, John Wiley, New York, 1989, Unit 13.10) including the popular one-step disruption, wherein a cloned DNA fragment containing the gene is digested with a restriction enzyme that cleaves the gene sequence, and another DNA fragment is cloned into the cleaved gene (Rothstein, R. J., Methods in Enzymology 101: 202-211 (1983)); integrative disruption, wherein an internal fragment of a cloned gene is introduced into the chromosome on an integrating plasmid, generating gene duplication of copies that are not intact so that a deletion in the chromosomal copy of the gene is generated; and clone transplacement, a rearrangement and replacement of DNA fragments accomplished by introducing a mutated, usually marked, gene on a plasmid, integrating it, allowing the plasmid to be evicted, and then screening for the mutant phenotype.
Sutter and Loper, cited above, for example, prepared mutants having disrupted cytochrome P450 reductase genes by replacing a 700 base pair cytochrome P450 reductase gene fragment with a 3.2 kilobase pair LEU2-containing fragment isolated from a plasmid to yield leucine proto-trophs denoted in the paper as TSY11-6C and TSY21 which contained disrupted cytochrome P450 reductase genes. Both strains were sensitive to ketoconazole. Another strain containing a disrupted cytochrome P450 reductase gene that is useful in this invention, denoted DCX45(pML82)-1B, is prepared by cloning the gene and disrupting it by insertion of the LEU2 yeast gene as described by Rothstein, cited above, and illustrated in Example 1 below.
A second strain of Saccharomyces cerevisiae that exhibits less sensitivity to compounds that inhibit cyto-chrome P450 is employed in the method of this invention. Strains exhibiting enhanced membrane permeability are preferred such as erg6 mutants described by Gaber, et al., and McCammon, M. T., et al., cited above, erg2, or erg5 mutants. As has been discussed, ERG6 is the putative structural gene for the enzyme that catalyzes the methylation at the C-24 position of sterol in the biosynthesis of ergosterol in yeast. Mutant erg6 strains have disrupted, deactivated or deleted ERG6 genes. Gaber, et al., prepared two erg6 mutants and found the gene to be not essential for growth, but the strains exhibited hypersensitivity to cycloheximide, resistance to nystatin, decreased mating frequency, decreased transformation freguency and decreased tryptophan uptake. One strain was a deletion-substitution mutant generated by deleting a 400 base pair in ERG6 and inserting a 1.9 kilobase pair fragment containing the LEU2 gene. A second strain was prepared by simply removing at least 90% (xcx9c2.9 kilobases) of the ERG6 gene. Any erg2, erg5 or erg6 mutant can be employed in the method of this invention. In one embodiment, erg6 mutant strain STX429-2A obtained from the Yeast Genetics Stock Center is employed.
In the practice of this invention""s method, the test sample is added to a culture or culture area of a Saccharomyces cerevisiae strain supersensitive to inhibitors of cytochrome P450 such as a strain having a disrupted cytochrome P450 reductase gene. At the same time, the test sample is added to a culture or culture area having a Saccharomyces cerevisiae strain exhibiting enhanced membrane permeability such as an erg6 mutant. The two cultures are incubated with the test samples together for such time under such conditions sufficient to observe yeast cell growth in corresponding cultures or culture areas of the strains containing no test sample. The extent of inhibition of growth in the culture or culture areas of the two strains are then compared. The presence of inhibition of cytochrome P450 or cytochrome P450 reductase is determined by observation of enhanced culture growth inhibition in the supersensitive strain.
Any type of solidified or liquid media that will support growth and reproduction of the S. cerevisiae strains may be employed as cultures in the method of this invention. Numerous yeast media are known to the skilled artisan, and an advantage of the invention is that baker""s yeast is relatively easy to grow. Typical media are yeast extract, peptone and dextrose (YEPD) or yeast extract and dextrose (YED) media; yeast basal growth media (YBGM) containing glucose, vitamins, minerals, and water; yeast extract, peptone, and adenine sulfate (YPA) media; yeast mannitol (YM) media and YM plus glucose; synthetic dextrose (SD) media containing dextrose, a nitrogen base, water, and, optionally amino acids, adenine sulfate and uracil; and the like.
Where liquid cultures are employed, differences in growth are generally determined by observing and comparing turbidity; for this purpose, optical density (OD) readings at 550 to 650 nm are made and compared. Preferred media, however, are solidified by adding agar or gelatin forming cultures in plates or dishes. Agar is especially preferred. In these embodiments, differential growth is observed visually.
In a preferred screening test, a Saccharomyces cerevisiae strain having a disrupted cytochrome P450 reductase gene such as TSY11-6C, TSY21 or DCX45(pML82)-1B is grown in culture in the presence of test samples. At the same time, a S. cerevisiae strain having an erg6 mutant such as STX429-2A is grown in culture with the same test samples.
Potentially active agents are identified by the observation of enhanced inhibition of the cytochrome P450 reductase disrupted strain over the erg6 strain.
In preferred embodiments, a positive control is employed to assist in the identification of potential agents. In these embodiments, a known inhibitor of a cytochrome P450 reductase related electron-transfer protein is employed. For example, a known lanosterol 14-xcex1-demethylase inhibitor such as ketoconazole, miconazole, dinaconazole, or econazole is useful as a control. Positive controls are added to cultures or culture areas of both S. cerevisiae strains, and the control effects on culture growth are compared to the cultures or culture areas with the test samples.
As mentioned above, particularly preferred embodiments employ solidified media, so that test samples and positive controls are observed visually and simultaneously as regions of the same culture. Samples or controls are introduced on a disk or in a well of the plate. Inhibition is observed visually as measurable zones around disks or wells in the lawn of growth in the plate or dish. Actives produce a much larger zone (xcx9cxe2x89xa78 mm) around test samples grown in a lawn of the reductase disrupted strain than in a lawn of the erg6 strain.
A distinct advantage of the invention is its speed and simplicity. The protocol is extremely simple. Many samples are readily analyzed in a short time, providing new potential sterol biosynthesis inhibitors that can be employed in the arsenal against undesirable fungi, some of which are resistant to currently known fungicides (including sterol biosynthesis inhibitors, see Koeller, W., and Scheinpflug, H., Plant Disease 71: 1066-1074 (1987)), and that interfere with pathogen but not host metabolism.
It is another advantage of the invention that it is sensitive, and only small amounts of biochemical or chemical agents are required for the test. In a standard assay of the invention, for example, which employs solidified media in a plate, as little as 20 xcexcg of a biochemical or chemical test sample or control are be applied to a disk or in a well.
The assay is a moderate positive rate assay (xcx9c0.04%), so that secondary tests may be considered to prioritize actives found using the screen. Standard in vitro and in vivo fungicide discovery screens can be employed for this. In vitro screens test samples for their ability to inhibit the growth of selected phytopathogenic fungi cultured in nutrient agar. These include but are not limited to fungi causing wheat eyespot (Pseudocercosporella herpotrichoides), rice s heath blight (Rhizoctonia solani) and damping off (Fusarium oxysporum), which all synthesize ergosterol. In in vivo screens, a variety of phytopathogenic fungi are used to infect plants treated with test compounds. Active compounds block or reduce the appearance of disease symptoms. A number of model plant infections can be employed and include ergosterol-producing fungi causing apple scab (Venturia inaequalis), pepper botrytis (Botrytis cincerea), rice blast (Pyricularia oryzae), sugar beet cercospora (Cercospora beticola), tomato early blight (Alternaria solani), wheat leaf rust (Puccinia recondita tritici), and wheat powdery mildew (Eerysiphe graminis tritici).
The following examples are p resented to further illustrate and explain the present invention and should not be taken as limiting in any regard.